Re: [ccp4bb] Phaser Fatal runtime error.
Data from Crysalis in mtz format are not merged I think - you have to go through the scale and merge step in Scala first ... Jan Dohnalek On Tue, Jun 26, 2012 at 11:21 PM, Steiner, Roberto roberto.stei...@kcl.ac.uk wrote: Don't know where the exact problem is. However, it is definitely possible to use a Crysalis-Scala-Truncate-Phaser pipeline without runtime errors. I have done a few times. I am sure you will be able to get help from Agilent people. If not, feel free to get back to me. Best Roberto On 26 Jun 2012, at 18:34, Stephen Carr wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.uk -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
Re: [ccp4bb] Phaser Fatal runtime error.
Dear Steve, The mtz file produced by the current version of CrysAlisPro is scaled but unmerged and requires SCALA to produce the merged data set. The new version of CrysAlisPro (currently under testing) will generate the merged mtz file as well. With regards, Tadeusz Agilent Technologies From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jan Dohnalek Sent: 27 June 2012 07:30 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Phaser Fatal runtime error. Data from Crysalis in mtz format are not merged I think - you have to go through the scale and merge step in Scala first ... Jan Dohnalek On Tue, Jun 26, 2012 at 11:21 PM, Steiner, Roberto roberto.stei...@kcl.ac.ukmailto:roberto.stei...@kcl.ac.uk wrote: Don't know where the exact problem is. However, it is definitely possible to use a Crysalis-Scala-Truncate-Phaser pipeline without runtime errors. I have done a few times. I am sure you will be able to get help from Agilent people. If not, feel free to get back to me. Best Roberto On 26 Jun 2012, at 18:34, Stephen Carr wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.ukmailto:stephen.c...@rc-harwell.ac.uk tel 01235 567717 Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.ukmailto:roberto.stei...@kcl.ac.uk -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
[ccp4bb] apply a spatial transformation on a pdb file n times
Dear colleagues, I want to apply a given x-y-z-rotation/transformation matrix on a pdb file, save the new pdb file and apply the same matrix again on the new pdb file and repeat this e.g. 100 times, thereby saving all pdb files (i.e. 01.pdb, 02.pdb, 03.pdb etc.). With pdbset this is very easy, but I'm wondering if there is any script to do so because I don't want to manually change file names all the time... I am not very familiar with scripting, but I found something in the internet. Can anybody help please with this i.e. is this a reasonable script to do so? # repeat a spatial transformation on a pdb file n times. #!/bin/csh -f start n=1 until n=x. pdbset xyzin mypdbfile($n).pdb xyzout mypdbfile($n+1).pdb eof-1 transform - 0.87831 0.47808 0 0 0 -1. -0.47808 0.87831 0 0.0 -2.713 0.0 eof-1 Thanks a lot, Chris DIPL.-ING. CHRIS FRÖHLICH Crystallography Dept. | Daumke group Max-Delbrück-Centre for Molecular Medicine (MDC) Robert-Rössle-Str. 10 13125 Berlin-Buch Lab: +49(0)30-9406-3263 Office: +49(0)30-9406-3275 Fax: +49(0)30-9406-3814 www.mdc-berlin.de/daumkehttp://www.mdc-berlin.de/daumke
Re: [ccp4bb] apply a spatial transformation on a pdb file n times
On 27/06/12 10:03, Froehlich, Chris wrote: Dear colleagues, I want to apply a given x-y-z-rotation/transformation matrix on a pdb file, save the new pdb file and apply the same matrix again on the new pdb file and repeat this e.g. 100 times, thereby saving all pdb files (i.e. 01.pdb, 02.pdb, 03.pdb etc.). With pdbset this is very easy, but I'm wondering if there is any script to do so because I don't want to manually change file names all the time... I am not very familiar with scripting, but I found something in the internet. Can anybody help please with this i.e. is this a reasonable script to do so? # repeat a spatial transformation on a pdb file n times. #!/bin/csh -f start n=1 until n=x. pdbset xyzin mypdbfile($n).pdb xyzout mypdbfile($n+1).pdb eof-1 transform - 0.87831 0.47808 0 0 0 -1. -0.47808 0.87831 0 0.0 -2.713 0.0 eof-1 In coot, you'd do it like this: (define m (list 0.87831 0.47808 0 0 0 -1. -0.47808 0.87831 0 0.0 -2.713 0.0)) (let ((orig (read-pdb start.pdb))) (for-each (lambda (n) (apply transform-molecule-by (cons orig m)) (write-pdb-file orig (string-append (number-string n) .pdb))) (range 100)))
[ccp4bb] Research Scientist position Munich
on behalf of Prof. Michael Sattler: JOB ADVERTISEMENT: The CRC 1035 “Conformational Control of Protein Function by conformational switching“ investigates conformational transitions of proteins and protein complexes using various biophysical and biochemical techniques. To complement existing expertise in X-ray crystallography, NMR spectroscopy and electron microscopy, Small Angle X-ray Scattering (SAXS) will be established at the Department Chemistry of the TU München. To support SAXS experiments, data analysis and the application in the research of the CRC 1035, a position for a Research Scientist is available, with a starting date as soon as possible and for an initial duration until June 30, 2016. Job description • Supervision and operation of a new table-top SAXS instrument • Support of users of the CRC 1035 for SAXS data acquisition and analysis • Implementation and development of SAXS methods for analysis of biological macromolecules for studies of conformation and dynamics in solution • Collaboration in multidisciplinary research projects • Interaction and collaboration with the Central Analytics Facilities at the TUM Department Chemie Requirements • Doctoral degree in physics, chemistry, biochemistry or related areas • Extensive research experience in the area of biological small angle scattering, preferably with a focus on soluble protein complexes: use of SAXS instrumentation, data acquisition and analysis • Independent and responsible work, highly motivated • Efficient communication and teamwork with an international team of scientists • Fluency in English and skills in computation and IT Salary is according to TV-L E13. Handicapped applicants will be given priority if equally qualified. TU München intends to increase the proportion of women; therefore applications of women are particularly welcome. http://www.nmr.ch.tum.de/aks/pdf/SFB1035_SAXS.pdf Please do not reply to me, send your application (CV, publications, research profile, references) via e-mail by July 23, 2012 to: Prof. Dr. Michael Sattler Lehrstuhl für Biomolekulare NMR-Spektroskopie Technische Universität München, Department Chemie Lichtenbergstr. 4, D-85747 Garching, Germany E-mail: waltraud.wolf...@helmholtz-muenchen.de
Re: [ccp4bb] apply a spatial transformation on a pdb file n times
Dear Chris, The script might work technically, but applying the same transformation matrix 100 times to the output of the previous iteration is a recipe for disaster. PDB files only store coordinates to 3 decimal places and your rounding errors will build up horribly. Far better to work out the transformation matrix/vector required for each iteration and apply that set to the original coordinates. Regards, Robert -- Dr. Robert Esnouf, University Research Lecturer and Head of Research Computing, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK Emails: rob...@strubi.ox.ac.uk Tel: (+44) - 1865 - 287783 and rob...@esnouf.comFax: (+44) - 1865 - 287547 Original message Date: Wed, 27 Jun 2012 09:03:13 + From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Froehlich, Chris chris.froehl...@mdc-berlin.de) Subject: [ccp4bb] apply a spatial transformation on a pdb file n times To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, I want to apply a given x-y-z-rotation/transformation matrix on a pdb file, save the new pdb file and apply the same matrix again on the new pdb file and repeat this e.g. 100 times, thereby saving all pdb files (i.e. 01.pdb, 02.pdb, 03.pdb etc.). With pdbset this is very easy, but I'm wondering if there is any script to do so because I don't want to manually change file names all the time... I am not very familiar with scripting, but I found something in the internet. Can anybody help please with this i.e. is this a reasonable script to do so? # repeat a spatial transformation on a pdb file n times. #!/bin/csh -f start n=1 until n=x. pdbset xyzin mypdbfile($n).pdb xyzout mypdbfile($n+1).pdb eof-1 transform - 0.87831 0.47808 0 0 0 -1. -0.47808 0.87831 0 0.0 -2.713 0.0 eof-1 Thanks a lot, Chris DIPL.-ING. CHRIS FRÃ*HLICH Crystallography Dept. | Daumke group Max-Delbrück-Centre for Molecular Medicine (MDC) Robert-Rössle-Str. 10 13125 Berlin-Buch Lab: +49(0)30-9406-3263 Office: +49(0)30-9406-3275 Fax: +49(0)30-9406-3814 www.mdc-berlin.de/daumke
Re: [ccp4bb] Phaser Fatal runtime error.
I suspect you didn't request the FreeR assignment on the TRUNCATE interface? This calls amongst other programs, CAD and CAD makes sure that the reflection list is unique and that it is in a standard asymmetric unit. Most people do it by default so don't hit these problems... I suggest you just run CAD (on the reflection utilities) and select - input all observations , then try again with the output mtz. All that will have happened is that the reflections are now assigned to a standard asymmetric unit and sorted. Eleanor On 26 Jun 2012, at 22:21, Steiner, Roberto wrote: Don't know where the exact problem is. However, it is definitely possible to use a Crysalis-Scala-Truncate-Phaser pipeline without runtime errors. I have done a few times. I am sure you will be able to get help from Agilent people. If not, feel free to get back to me. Best Roberto On 26 Jun 2012, at 18:34, Stephen Carr wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.uk
Re: [ccp4bb] Phaser Fatal runtime error.
Just to be clear, Phaser does not require the reflections to be in a standard asymmetric unit, only that they are unique (as the error message thrown says). Airlie On Jun 27 2012, Eleanor Dodson wrote: I suspect you didn't request the FreeR assignment on the TRUNCATE interface? This calls amongst other programs, CAD and CAD makes sure that the reflection list is unique and that it is in a standard asymmetric unit. Most people do it by default so don't hit these problems... I suggest you just run CAD (on the reflection utilities) and select - input all observations , then try again with the output mtz. All that will have happened is that the reflections are now assigned to a standard asymmetric unit and sorted. Eleanor On 26 Jun 2012, at 22:21, Steiner, Roberto wrote: Don't know where the exact problem is. However, it is definitely possible to use a Crysalis-Scala-Truncate-Phaser pipeline without runtime errors. I have done a few times. I am sure you will be able to get help from Agilent people. If not, feel free to get back to me. Best Roberto On 26 Jun 2012, at 18:34, Stephen Carr wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.uk
[ccp4bb] hi all
Hi all , thank you for being new member in this group. i also just stated with my PhD in structural biology at Hamburg University (DESY) . i have no Idea about crystallography . I just go step by step. so please help me to find my way for excellence in structural biology. i appreciate your suggestion like books, review articles, advices,lectures, power point presentation but more simple . thanks a lot for reading yours Amr
Re: [ccp4bb] Phaser Fatal runtime error.
We have an in-house Agilent (Oxford) system and routinely use data with CCP4. You will need to run sortmtz, scala (w/constant scale), and truncate to prep the data properly. This can be done via batch file or GUI.You may also have to reset/reassign the space group for some space groups due to an apparent bug in the CrysalisPro MTZ conversion routine. You can find details at capsicum.colgate.edu/chwiki in our crystallography pages. Roger Rowlett On Jun 26, 2012 1:34 PM, lt;Stephen Carrgt; stephen.c...@rc-harwell.ac.uk wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717
Re: [ccp4bb] hi all
Your question is way to broad to be answered in a reasonable time/space. As for books (plenty of options exist beyond these) There is a 1976 classic http://www.amazon.com/Protein-Crystallography-Molecular-Biology-Series/dp/0121083500 And of course there is a more recent highly recommended http://www.ruppweb.org/Garland/ On a more general note (and this is *not* to fault you for asking), aren't you supposed to be getting advise of this kind from your PhD advisor? I presume that she/he is a structural biologist and undoubtedly qualified to guide a graduate student. Cheers, Ed. On 06/27/2012 07:34 AM, amro selem wrote: Hi all , thank you for being new member in this group. i also just stated with my PhD in structural biology at Hamburg University (DESY) . i have no Idea about crystallography . I just go step by step. so please help me to find my way for excellence in structural biology. i appreciate your suggestion like books, review articles, advices,lectures, power point presentation but more simple . thanks a lot for reading yours Amr -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] hi all
Hi, Two educational softwares which might help you (off course in conjunction with books and articles) are 1) XrayView 2)SpacegroupViz Best Arko On Wed, Jun 27, 2012 at 6:22 PM, Ed Pozharski epozh...@umaryland.eduwrote: Your question is way to broad to be answered in a reasonable time/space. As for books (plenty of options exist beyond these) There is a 1976 classic http://www.amazon.com/Protein-Crystallography-Molecular-Biology-Series/dp/0121083500 And of course there is a more recent highly recommended http://www.ruppweb.org/Garland/ On a more general note (and this is *not* to fault you for asking), aren't you supposed to be getting advise of this kind from your PhD advisor? I presume that she/he is a structural biologist and undoubtedly qualified to guide a graduate student. Cheers, Ed. On 06/27/2012 07:34 AM, amro selem wrote: Hi all , thank you for being new member in this group. i also just stated with my PhD in structural biology at Hamburg University (DESY) . i have no Idea about crystallography . I just go step by step. so please help me to find my way for excellence in structural biology. i appreciate your suggestion like books, review articles, advices,lectures, power point presentation but more simple . thanks a lot for reading yours Amr -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
Re: [ccp4bb] The effect of His-tag location on crystallization
Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
Re: [ccp4bb] The effect of His-tag location on crystallization
With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
[ccp4bb] A postdoctoral position in Dr. Yongqun Zhu lab in LSI, Zhejiang University
A postdoctoral position is available in the laboratory of Dr. Yongqun Zhu in Life Sciences Institute, Zhejiang University. Our lab interest is to take approaches of structural biology to study the pathogen-host interactions and cellular signaling transduction involved in cancer biology. In addition to structural biology, our lab is also actively involved in functional studies. The applicant should have or will have a Ph.D of structural biology, biochemistry or cell biology. A highly motivated candidate with strong background in structural biology or biochemistry is encouraged. Prior experience in X-ray crystallography is desirable, but not essential. Life Sciences Institute located in Hangzhou, a beautiful city in China, is fully funded by Zhejiang University and aims to be a worldwide first-class research institute (http://lsi.zju.edu.cn/). Our lab is well-equipped with state of art instrumentations for structural and functional studies. An internationally competitive salary and benefit package for the postdoctoral position will be provided. To apply, please email a CV and at least two recommendation letters to Dr. Yongqun Zhu (zhuyong...@zju.edu.cn or zhuyong...@yahoo.com.cn ). Yongqun Zhu, Ph.D Professor, Life Sciences Institute, Zhejiang University
[ccp4bb] PhD Studentship in Dundee, UK
A PhD studentship is available in Dundee to work in the laboratory of Bill Hunter. The project involves the application of crystallographic methods to support early stage drug discovery targeting bacterial pathogens. A series of targets are available and the student will be engaged in protein purification, structural studies and the application of screening methods to search out novel ligands and inhibitors. The lab in Dundee is well equipped for all aspects of the research. Some experience with membrane bound proteins would be an advantage. The funding, which is available immediately, can only support a student holding a european passport and an honours degree, first or upper second, in a relevant subject. Please send a copy of your cv, names and contact details of three referees to Bill hunter (w.n.hun...@dundee.ac.ukmailto:w.n.hun...@dundee.ac.uk). The University of Dundee is a registered Scottish Charity, No: SC015096
Re: [ccp4bb] The effect of His-tag location on crystallization
I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice pr...@uchicago.edu wrote: With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
Re: [ccp4bb] The effect of His-tag location on crystallization
Here's another example: http://www.pdb.org/pdb/explore/explore.do?structureId=2F62 dimer with His-tag-ears without His6-tag this would not have been possible. Jürgen On Jun 27, 2012, at 12:04 PM, Brad Bennett wrote: I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice pr...@uchicago.edumailto:pr...@uchicago.edu wrote: With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723tel:773%20834%201723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724tel:%28517%29%20355-9724 Lab: (517) 353-9125tel:%28517%29%20353-9125 FAX: (517) 353-9334tel:%28517%29%20353-9334 Email: rmgarav...@gmail.commailto:rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] The effect of His-tag location on crystallization
Or indeed support surreal structures :-))
(PMID: 11853672 vs PMID: 14749821 and so on)
--
A. Radu Aricescu, PhD
University Research Lecturer
MRC Career Development Award Fellow
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive, Oxford OX3 7BN
United Kingdom
Phone: +44-1865-287564
Fax: +44-1865-287547
Original message
Date: Wed, 27 Jun 2012 12:21:22 -0400
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Bosch,
Juergen jubo...@jhsph.edu)
Subject: Re: [ccp4bb] The effect of His-tag location on crystallization
To: CCP4BB@JISCMAIL.AC.UK
Here's another example:
http://www.pdb.org/pdb/explore/explore.do?structureId=2F62
dimer with His-tag-ears without His6-tag this
would not have been possible.
Jürgen
On Jun 27, 2012, at 12:04 PM, Brad Bennett wrote:
I think it was an N-terminal RGS-type His tag in
3O8Y (human lipoxygenase) that mediated crystal
contacts with a symmetry related molecule. As I
recall, this tag composed a B-strand that formed a
nice interface with a native B-strand of the
symmetry related molecule. Pretty cool...
-Brad
On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice
pr...@uchicago.edu wrote:
With Flp recombinase - DNA complexes, a
C-terminal His tag triggered a different (but
sadly not better) crystal form, and the His side
chains packed against the bases at the end of a
neighboring DNA duplex.
=
Phoebe A. Rice
Dept. of Biochemistry Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.rsc.org/shop/books/2008/9780854042722.asp
Original message
Date: Wed, 27 Jun 2012 10:14:58 -0400
From: CCP4 bulletin board
CCP4BB@JISCMAIL.AC.UK (on behalf of R. M.
Garavito rmgarav...@gmail.com)
Subject: Re: [ccp4bb] The effect of His-tag
location on crystallization
To: CCP4BB@JISCMAIL.AC.UK
Most of the comments you will get will be
anecdotal
in that people will report the successful
results
and do not take the time or effort to
characterize
the less successful results. This often
occurs
because the tagged portion of the protein is
most
often disordered, even in the best crystals.
Thus,
other than saying tagging on this end
works, but
tagging on that end doesn't, there is
little more
you can say. Each case will be different,
and it is
almost impossible to arrive at any
generalized
conclusion.
We prefer C-terminal tagged proteins for a
number of
reasons, but if an N-terminally tagged
protein
crystallizes well, so be it. Of the dozens
of N-
and C-tagged protein structures we have
solved in my
lab and with collaborators, I have only seen
one
case of an ordered His-tag: the His
residues had
coordinated Cd ions, which proved essential
for
getting good crystals. However, beyond that
there
was not much more to say.
For your protein and the resulting crystals,
an
N-terminally tagged protein crystallized
well.
Whether you can draw any more conclusions
from
these results depends on characterizing
crystals of
both N- and C-tagged proteins. Just
assuming that
the C-tagged protein is trying to
crystallize in the
same or related crystal form as the N-tagged
protein
is an unwarranted assumption without
experimental
evidence to back it up. That is why most
groups
just run with the winner.
Cheers,
Michael
R. Michael Garavito, Ph.D.
Professor of Biochemistry Molecular
Biology
603 Wilson Rd., Rm. 513
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517)
353-9125
FAX: (517) 353-9334
Email: rmgarav...@gmail.com
On Jun 26, 2012, at 9:06 PM, weliu wrote:
Dear all,
We crystallized a protein and found that
crystal
quality greatly depended on the location
of
His-tag. When a His-tag was added at the
C-terminus, only crystalline
Re: [ccp4bb] The effect of His-tag location on crystallization
Human leukotriene C4 synthase (PDB accession code: 2UUI) is another example, illustrating how an N-terminal polyhistidine-tag, in conjunction with metals, presumably facilitated crystallization. On Jun 27, 2012, at 12:04 PM, Brad Bennett wrote: I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice pr...@uchicago.edu wrote: With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
Re: [ccp4bb] relative intensity of ice rings
I thank for the references and the comprehensive discussion from Dr. Holton. Also, for the reference indicated by Dr. Berry. I think I will get what I am looking for, now I need to process all this information. Partially answering Dr. Holton, my aim is to have a side guide for improving parameters to process images with ice diffraction rings. In general, the idea is to exclude the minimum area from the detector, but large enough to avoid bad regions. In this, ice ring widths have a role, so, besides the amount of ice, exposure time and beam intensity, relative diffraction intensities contribute, and this way one could decide better what the best width might be for each individual ring. Sure, looking at the images are the base here, but it is interesting to be seconded by the ring expected positions and relative intensities. Yours, Jorge On Tuesday 26 June 2012 09:16 PM, James Holton wrote: I think an appropriate reference is Bragg (1921) http://dx.doi.org/10.1088/1478-7814/34/1/322 who compared various possible crystal structures to the relative strength of the reflections from ice powder measured by Dennison (1921) http://dx.doi.org/10.1103/PhysRev.17.20. However, as Bragg noted Dennison's intensities don't agree all that well with those you would expect from what we now know is the structure of hexagonal ice (Ih). It is possible that Dennison's preparation (plunge-cooling pure water in a capillary) actually created some cubic ice (ice Ic) along with his hexagonal ice (ice Ih). The high intensity he saw for the middle line of the triplet we normally see for true hexagonal ice is consistent with this. Cubic ice is actually more commonly seen in MX diffraction patterns than hexagonal ice (in my experience). However, you do have to be very careful about what you mean by intensity. Are you talking about photons/pixel? Photons/spot? Photons integrated over a powder_ring? All these will be different relative numbers. I'm not sure if they knew about Lorentz factors yet in 1921 There is no mention of correcting for them in either paper. Anyway, if you are after the true hexagonal ice ring intensities, I would advise taking the following PDB file: CRYST14.5114.5117.346 90.00 90.00 120.00 P 63/m m c SCALE1 0.221680 0.127987 -0.00 -0.0 SCALE2 -0.00 0.255974 -0.000.0 SCALE3 0.00 -0.00 0.136129 -0.0 ATOM 1 O WAT A 1 0.000 2.604 0.457 1.00 0.00 O ANISOU1 O WAT A 1 603630172302 0 0 O ATOM 2 H WAT A 1 0.000 2.604 1.308 0.50 0.00 H ANISOU2 H WAT A 1 510510 56255 0 0 H ATOM 3 H WAT A 1 0.000 3.432 0.148 0.50 0.00 H ANISOU3 H WAT A 1 487361163185199 95 H Calculate structure factors from it, add up F2 of same-resolution indicies and plot them out that way. remember, the square of a structure factor is proportional to the integrated intensity of a single-crystal spot, which is not the same thing as a powder ring intensity. The relationship was described most recently by Norby (1997) http://dx.doi.org/10.1107/S0021889896009995 Which I paraphrase as: for a flat detector, the average intensity of a pixel in a powder ring is given by: Ipix = k*p*sum(F2)*omega/sin(theta)/sin(2*theta) where Ipix is the recorded value of one pixel, 'k is a resolution-independent scale factor, p is the polarization factor (see Holton Frankel 2010), F is the structure factor of an hkl index that falls on the ring (there can be more than one, hence the sum), omega is the solid angle subtended by the pixel and theta is the Bragg angle. For the above PDB, I get: d sum(F2) 3.907 121 3.673 156 3.449 111 2.676 88.5 2.255 111 2.075 153 1.953 62.9 1.922 84.2 1.888 62.5 1.836 4.33 1.725 47.8 1.662 1.84 1.527 96.7 1.477 42.8 1.448 39.5 1.375 78.9 1.370 34.6 HTH, -James Holton MAD Scientist On Tue, Jun 26, 2012 at 2:34 PM, Edward A. Berryber...@upstate.eduber...@upstate.edu wrote: Maybe figure 4 in Viatcheslav Berejnov et al. Vitrification of aqueous solutions J. Appl. Cryst. (2006). 39, 244–251 ? JORGE IULEK wrote: Hi, all, I thought I could easily find a reference to comment upon the relative intensity of rings in an image due to diffraction by polycrystal ice, but no luck googling for that. A reference that would contain a picture (with visual relative intensities) would be even better. Of course absolute intensity depends on the amount of ice, but a relative scale is what I am looking for now. Yours, Jorge
Re: [ccp4bb] The effect of His-tag location on crystallization
You may want to have a look at 3ur7 and 3ur8. I have also another example of a glycohydrolytic enzyme with an N-terminal His-tag sitting in the active site of a symmetry-related molecule (not deposited yet). Karolina On 27/6/2012, Brad Bennett bradbennet...@gmail.com wrote: I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice pr...@uchicago.edu wrote: With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
Re: [ccp4bb] relative intensity of ice rings
It is better to spent time learning how to collect without ice… :-) FF Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Jun 27, 2012, at 21:00 , JORGE IULEK wrote: I thank for the references and the comprehensive discussion from Dr. Holton. Also, for the reference indicated by Dr. Berry. I think I will get what I am looking for, now I need to process all this information. Partially answering Dr. Holton, my aim is to have a side guide for improving parameters to process images with ice diffraction rings. In general, the idea is to exclude the minimum area from the detector, but large enough to avoid bad regions. In this, ice ring widths have a role, so, besides the amount of ice, exposure time and beam intensity, relative diffraction intensities contribute, and this way one could decide better what the best width might be for each individual ring. Sure, looking at the images are the base here, but it is interesting to be seconded by the ring expected positions and relative intensities. Yours, Jorge On Tuesday 26 June 2012 09:16 PM, James Holton wrote: I think an appropriate reference is Bragg (1921) http://dx.doi.org/10.1088/1478-7814/34/1/322 who compared various possible crystal structures to the relative strength of the reflections from ice powder measured by Dennison (1921) http://dx.doi.org/10.1103/PhysRev.17.20. However, as Bragg noted Dennison's intensities don't agree all that well with those you would expect from what we now know is the structure of hexagonal ice (Ih). It is possible that Dennison's preparation (plunge-cooling pure water in a capillary) actually created some cubic ice (ice Ic) along with his hexagonal ice (ice Ih). The high intensity he saw for the middle line of the triplet we normally see for true hexagonal ice is consistent with this. Cubic ice is actually more commonly seen in MX diffraction patterns than hexagonal ice (in my experience). However, you do have to be very careful about what you mean by intensity. Are you talking about photons/pixel? Photons/spot? Photons integrated over a powder_ring? All these will be different relative numbers. I'm not sure if they knew about Lorentz factors yet in 1921 There is no mention of correcting for them in either paper. Anyway, if you are after the true hexagonal ice ring intensities, I would advise taking the following PDB file: CRYST14.5114.5117.346 90.00 90.00 120.00 P 63/m m c SCALE1 0.221680 0.127987 -0.00 -0.0 SCALE2 -0.00 0.255974 -0.000.0 SCALE3 0.00 -0.00 0.136129 -0.0 ATOM 1 O WAT A 1 0.000 2.604 0.457 1.00 0.00 O ANISOU1 O WAT A 1 603630172302 0 0 O ATOM 2 H WAT A 1 0.000 2.604 1.308 0.50 0.00 H ANISOU2 H WAT A 1 510510 56255 0 0 H ATOM 3 H WAT A 1 0.000 3.432 0.148 0.50 0.00 H ANISOU3 H WAT A 1 487361163185199 95 H Calculate structure factors from it, add up F2 of same-resolution indicies and plot them out that way. remember, the square of a structure factor is proportional to the integrated intensity of a single-crystal spot, which is not the same thing as a powder ring intensity. The relationship was described most recently by Norby (1997) http://dx.doi.org/10.1107/S0021889896009995 Which I paraphrase as: for a flat detector, the average intensity of a pixel in a powder ring is given by: Ipix = k*p*sum(F2)*omega/sin(theta)/sin(2*theta) where Ipix is the recorded value of one pixel, 'k is a resolution-independent scale factor, p is the polarization factor (see Holton Frankel 2010), F is the structure factor of an hkl index that falls on the ring (there can be more than one, hence the sum), omega is the solid angle subtended by the pixel and theta is the Bragg angle. For the above PDB, I get: d sum(F2) 3.907 121 3.673 156 3.449 111 2.676 88.5 2.255 111 2.075 153 1.953 62.9 1.922 84.2 1.888 62.5 1.836 4.33 1.725 47.8 1.662 1.84 1.527 96.7 1.477 42.8 1.448 39.5 1.375 78.9 1.370 34.6 HTH, -James Holton MAD Scientist On Tue, Jun 26, 2012 at 2:34 PM, Edward A. Berryber...@upstate.edu wrote: Maybe figure 4 in Viatcheslav Berejnov et al. Vitrification of aqueous solutions J. Appl. Cryst. (2006). 39, 244–251 ? JORGE IULEK wrote: Hi, all, I thought I could easily find a reference to comment upon the relative intensity of rings
